1. Field of the Invention
The present invention relates to a new process for effecting the acylation step in amide formation, especially in peptide synthesis.
2. Description of the Prior Art
Polypeptides, especially proteins, play a critical role in fundamental biochemical processes in living cells. Biochemical reactions, including metabolic reactions, are catalyzed by enzymes, which are comprised of proteins. These proteins are chiral molecules, and it is often the case that of the various stereoisomers that may possibly exist, one is usually the most efficacious.
Moreover, polypeptides are useful as medicaments. In recent years, peptides have been found useful in combating various diseases, including cancer, diabetes, plant toxins, and the like. Additionally, peptides have shown specific activity as growth promoters, suppressants, antibiotics, insecticides, contraceptives, anti-hypertensives, sleep inducers, anti-depressants, analgesics, and so on.
The synthesis of proteins has always been a challenge to chemists. However, chemical synthesis offers advantages not realized by genetic engineering and other biological approaches such as isolation of natural proteins. First, it is useful in confirming the structure of a protein. Moreover, protein synthesis is necessary to synthesize analogs, allowing scientists to evaluate biological activity and/or pharmacological efficacy in relation to molecular structure.
Success in the chemical synthesis of peptides relies, in part, on the use of the appropriate coupling reagents in combination with the appropriate protecting groups. Especially in peptide synthesis, formation of the peptide bond between two amino acids requires activation of the carboxyl group of one of the amino acids before the reaction can occur. However, the activation step in conjunction with the coupling reaction causes a serious problem of loss of configuration at the carboxyl residue which has been activated. Thus, in designing chemical syntheses of peptides, the objective is to provide the peptide product in good yield and maintenance of the configurational integrity of the carboxylic component, i.e., minimal racemization. Thus, the duality of good yield and minimal or no racemization is difficult to achieve because the best methods require the acid to be converted to a derivative bearing a good leaving group. Thus, under normal coupling conditions, there is a loss of configuration.
Moreover, current methods of syntheses also tend to produce side reactions which decrease yield.
Currently, syntheses of peptides are in solution by classical or various repetitive methods. Alternatively, peptides may be prepared on a solid support (Merrifield method). These are all popular techniques in synthesizing peptides from the coupling of two or more amino acids, in synthesizing larger peptides from the coupling of amino acids with smaller peptides or in the coupling of smaller peptides. Solution methods have the advantage of being easily monitored, allowing purification of intermediates, if necessary, at any stage. A major drawback, however, is the relative slow pace of synthesis, with each step being carried out manually.
The major advantage of the Merrifield method is its easy automation so that unattended, computer-controlled machine synthesis is possible. Unfortunately, the method suffers from an inherent deficiency due to the insoluble nature of the support on which the synthesis proceeds. Unless each acylation step occurs with approximately 100% efficiency, mixtures will inevitably be built up on the polymer. The longer the chain, the greater will be the contamination by undesired side reactions. Side products produced in such reactions remain to contaminate the desired product when it is removed from the polymeric matrix at the end of the cycle. These current techniques are not useful in preparing peptides of greater than 40-50 residues; separation of side products from the desired product becomes increasingly difficult when larger peptides are synthesized.
For very long segments (50 or more amino acids), therefore, current methods are not satisfactory. Often, mixtures are obtained of such forbidding complexity that it may be difficult or impossible to isolate the desired peptide.
The problems enumerated hereinabove may be eliminated if the proper derivatives of the underlying amino acids and/or the proper conditions for the coupling reaction could be found. Protecting groups, such as t-butyloxycarbonyl (t-Boc) or N-α-(9-fluorenylmethyl)oxycarbonyl (Fmoc), have been used to minimize side reactions.
The most commonly used coupling reagents are carbodiimides such as dicyclohexylcarbodiimides, diisopropylcarbodiimides, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimides used with various additives.
Additives generally inhibit side reactions and reduce racemization. Heretofore, the most common peptide coupling additive used during peptide coupling for peptide synthesis is 1-hydroxybenzotriazole (HOBt). This reagent has been used either in combination with a carbodiimide or other coupling agent or built into a stand alone reagent, such as 1-benzotriazolyoxytris (dimethylamino)phosphonium hexafluorophosphate (BOP) or an analogous uronium salt. HOBt is applicable to both stepwise and segment condensations. However, many cases have been encountered in which HOBt is ineffective, possibly because of steric effects, or low basicity of the amino component. Especially problematic are segment couplings at amino acid units other than glycine or proline, since the problem of racemization may be severe. The related N-hydroxybenzotriazinone (HOOBt) may provide better protection against racemization, but it is rarely used due to competing side reactions involving ring openings. A drawback in the use of BOP is that it produces a toxic side product, hexamethylphosphorotriamide.
Recently other coupling reagents have been introduced, such as N-[1H-benzotriazo-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HBTU), N-[(1-H-benzotriazol)dimethylamino)methylene]N-methylmethanaminium tetrafluoroborate N-oxide (TBTU), O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethylene uranium hexafluorophosphate (HBMDU), O-(benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate (HBPyU) and O-(benzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uronium hexafluorophosphate (HBPipU).
Another additive that has been used in peptide synthesis is 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt). HODhbt has proved to be generally superior to HOBt. Moreover, its use permits one to follow the completion of the reaction visually by a color change which occurs when acylation is complete. However, HODhbt has problems associated therewith due to inherent side reactions.
Other derivatives, which include O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,1-tetra-methyluronium tetrafluoroborate and [3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)oxy]tris(pyrrolidino)phosphonium hexafluorophosphate also have applications in peptide coupling.
Other reagents for facilitating peptide coupling have also been described. For example, in Tetrahedron Letters, 1994, 2279-2282, Carpino, et al. disclose that 1-hydroxy-7-azabenzotriazole and its corresponding uronium and phosphonium salts, designated HAPyU and AOP, respectively, were effective in promoting peptide coupling and avoiding racemization in a model solid-phase peptide segment coupling process. In addition, Carpino, et al. disclose in J. Org. Chem., 1994, 59, 695-698 that azabenzotriazolyluronium salts, e.g., designated as HBTU, HATU, HAPyU, and HAMDU, are useful in peptide synthesis.
U.S. Pat. No. 5,644,029 to Carpino discloses, among other things, the use of compounds of the following formula in promoting peptide coupling:
or N-oxides thereof or salts thereof wherein
R1 and R2 taken together with the carbon atoms to which they are attached form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic containing from 3 and up to a total of 13 ring carbon atoms, said heteroaryl may be unsubstituted or substituted with lower alkyl or an electron-donating group;
Y is O, NR4, CR4R5;
R5 is independently hydrogen or lower alkyl;
X is CR6R7 or NR6;
R6 and R7 are independently hydrogen or lower alkyl; or R6 and R7 taken together form an oxo group or when n=0, R4 and R6 taken together may form a bond between the nitrogen or carbon atom of Y and the nitrogen or carbon atom of X;
Q is (CR8R9) or (NR8);
when n is 1, R4 and R8 taken together may form a bond between the ring carbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R8;
n is 0 or 1;
R3 is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl,
a positively charged electron withdrawing group, SO2R14, or

R14 is lower alkyl, aryl or lower arylalkyl; q is 0-3;
R8 and R9 are independently hydrogen or lower alkyl or R7 and R8 taken together with the carbon to which they are attached form an aryl ring, AA1 is an amino acid and BLK is an amino protecting group, and m is 0 or 1.
The present inventor has found other coupling agents, which provide relatively pure products with little, if any, side products being co-produced and minimal, if any, racemization. Moreover, the reaction conditions are very mild and the reagents used are easy to prepare. Thus, by using the compounds of the present invention as additives, the yield of the peptide s enhanced and little, if any, racemization occurs.